NAD metabolism is rapidly altered following genotoxic stress by the activation of ADP-ribose (ADPR) polymer cycles. These cycles are mediated by poly(ADP-ribose) polymerases 1 and 2 (PARP-1, PARP-2) and poly ADP-ribose glycohydrolase (PARG) and they play a major role in the maintenance of genomic integrity by mediating cell recovery or cell death following genotoxic stress. PARG functions in the nucleus where ADPR polymers are synthesized but we have discovered a mitochonddal isoform of PARG. The hypothesis to be tested is that nuclear and mitochondrial PARG are key components of a signaling and sensing mechanism reporting the amount of DNA damage and integrating this information with key mitochondrial regulators of cell death. The presence of PARG in mitochondria is intriguing as PARP-1 initiated ADPR polymer cycles are linked to release of apoptosis inducing factor (AIF) from mitochondria following genotoxic stress but PARP-1 itself is not present in mitochondria. Our hypothesis predicts that nuclear PARG promotes cell recovery and prevents cell death by preventing formation of free ADPR polymers at low levels of genotoxic stress but generates free ADPR polymers that can be released from the nucleus following high levels of genotoxic stress to participate in AIF release from mitochondria. The mitochondrial PARG isoform prevents inappropriate AIF release by ADPR polymers and must be saturated with ADPR polymers before AIF can be released from mitochondda. The specific aims by which this hypothesis will be tested include defining the role of nuclear and mitochonddal PARG isoforms in DNA damage response signaling leading to cell recovery and cell death (Aim 1), defining the regulation of PARG gene expression and the relationship between the expression of the PARG gene and the mitochondrial import protein gene TIM23 with which it shares a common promoter (Aim 2), and defining key structural features of the PARG protein (Aim 3). We will modulate PARG content and activity using PARG gene disrupted mouse cells, novel cell permeable PARG inhibitors, and by overexpression PARG isoforms and site-directed mutants. The innovation of the application is that is represents a new paradigm for cross talk between nucleus and mitochondria for regulation of cell death and that powerful genetic and chemical tools have been developed to test the central hypothesis. The proposed research also provides the opportunity to evaluate PARG as a therapeutic target.